Abstract/ Summary Recent studies assessing the immune phenotypes and functionality of septic patients have increased our understanding of the immune dysregulation that occurs during sepsis. The new paradigm suggests that the balance of T cell costimulatory and coinhibitory molecules is critical to determining the outcome of T cell activation and/or dysregulation during sepsis. One costimulatory molecule that is most critical to T cell activation is CD28. CD28 is a potent costimulatory receptor and on nave T cells is a required secondary signal that promotes optimal T cell activation. CD28 has been shown to be down-regulated during sepsis on CD4+ and CD8+ T cells in both mice and humans. These expression data have led to the supposition that CD28 down-regulation may contribute to immune suppression following sepsis. However, the functional role of CD28 during sepsis is controversial, as other studies have shown a protective effect of CD28 blockade in several murine models of sepsis. Of note, CD28 is differentially expressed on nave vs. memory T cells. Moreover, even memory T cells which retain CD28 expression may have reduced dependence on CD28 signaling for activation. This is important because memory T cells constitute roughly 50% of the human T cell compartment by the time most people reach adulthood but are much less frequent (<5%) in laboratory mice. Thus, we hypothesized that the conflicting reports and unclear role of CD28 during sepsis may be due to a differential role on memory vs. nave T cells. In order to test this hypothesis and better model the antigen-experienced immune system of human patients, during the last funding cycle we developed a model to generate mice that possess a memory T cell compartment more similar to that of adult humans, that could then be rendered septic. This work was recently published in JCI Insight. Briefly, nave animals were sequentially infected with different acutely cleared bacterial and viral infections. This approach generates a mouse with ~20% CD4+ and ~60% CD8+ memory T cells, but the actual infections are completely resolved and no virus/ bacteria are detectable beyond day ~38, thus mitigating any impact of persistent infection. Mice then undergo cecal ligation and puncture (CLP). Compellingly, our preliminary data reveal a striking but opposite effect of CD28 blockade on sepsis-induced mortality in the nave vs. memory models. While treatment of nave laboratory animals with anti-CD28 resulted in an increase in mortality, treatment of antigen-experienced ?memory mice? with anti- CD28 resulted in a significant decrease in mortality as compared to untreated memory controls. Here, we propose to interrogate the mechanisms by which inhibition of CD28-mediated costimulatory signals protects ?memory mice? but not nave laboratory animals from death during sepsis. These studies are significant and highly clinically relevant because they will facilitate the development of immunomodulatory strategies to target the CD28 pathway on physiologically relevant cell types in order to prevent death in septic human patients.